Method for producing a hydroxylation catalyst and the use thereof

ABSTRACT

A process for preparing a hydroxylation catalyst by i) embedding a cystochrome P450 monooxygenase in a sol-gel matrix, ii) embedding an enzymatic NADPH-regenerating system in a sol-gel matrix, and combining the two components i) and ii) unless they were already mixed together before the embedding.

The present invention relates to a process for preparing a hydroxylationcatalyst based on a cytochrome P450 monooxygenase, and to processes forthe hydroxylation of organic substrates using these hydroxylationcatalysts.

Cytochrome P450 monooxygenases (called CYP hereinafter) catalyze thehydroxylation of a number of hydrophobic substrates at activated andnonactivated carbon atoms. This involves one oxygen atom of O₂ beingincorporated into the substrate, while the other oxygen atom is reducedto H₂O with simultaneous oxidation of nicotine adenine dinucleotide(phosphate) (NAD(P)H). This hydroxylation proceeds regio- andstereospecifically in many cases.

A particularly promising cytochrome P450 monooxygenase is the geneoriginally cloned from Bacillus megaterium, called CYP BM-3 hereinafter.This is a natural fusion protein with a size of 119 kDa and composed ofa heme-comprising monooxygenase domain and an FAD- and FMN-comprisingreductase domain (L. P. Wen, A. J. Fulco, J. Biol. Chem. 1987, 262,6676-6682; A. J. Fulco, R. T. Ruettinger, Life Sci. 1987, 40,1769-1775).

The natural substrates for CYP BM-3 are long-chain fatty acids(C12-C20), which are hydroxylated exclusively at the subterminalpositions ω-1, ω-2, ω-3, in some cases with high enantioselectivity.Variants of CYP BM-3 (called muteins) also show activity towardunnatural substrates such as short-chain fatty acids (Ost et al. FEBSLett. 2000, 486, 173-177; Li et al. Biochim. Biophys. Acta 2001, 1545,114-121), indoles (Li et al. Chemistry 2000, 6, 1531-1536), polycyclicaromatic hydrocarbons (Li et al. Appl. Environ. Microbiol. 2001, 67,5735-5739), alkanes (Appel et al. J. Biotechnol. 2001, 88, 167-171) andstyrenes (Li et al. FEBS Lett. 2001, 508, 249-252).

Use of this CYP on the industrial scale is still impeded by the problemsof low stability and product removal. A further disadvantage is thedependence of CYP on costly cofactors such as NADPH.

These problems can be solved in part by employing immobilized CYP forthe hydroxylation reactions. However, many immobilization processes atleast partly inactivate the enzyme, or the diffusion-controlled supplyof necessary cofactors and substrates to the enzyme is limited.

It is an object of the present invention to provide a process whichpermits CYP to be immobilized with high enzymatic activity.

The invention relates to a process for preparing a hydroxylationcatalyst by

i) embedding a cytochrome P450 monooxygenase in a sol-gel matrix,

ii) embedding an enzymatic NADPH-regenerating system in a sol-gelmatrix,

-   -   and combining the two components i) and ii) unless they were        already mixed together before the embedding.

Cytochrome P450 monooxygenases (CYP) and their use in biotransformationsare known to the skilled worker for example from E. T. Farinas et al.Adv. Synth. Catal. 2001, 343, 601-606 or V. Urlacher and R. D. SchmidCurr. Opin. Biotechnol. 2002, 13, 557-564.

CYP which are particularly well suited for the process of the inventionare those originally isolated from microorganisms, especially those ofthe genus Bacillus.

A particularly suitable cytochrome P450 monooxygenase is the proteinoriginally cloned from Bacillus megaterium, called CYP BM-3 hereinafter.This is a natural fusion protein with a size of 119 kDa and composed ofa heme-comprising monooxygenase domain and an FAD- and FMN-comprisingreductase domain (L. P. Wen, A. J. Fulco, J. Biol. Chem. 1987, 262,6676-6682; A. J. Fulco, R. T. Ruettinger, Life Sci. 1987, 40,1769-1775).

It is possible starting from this CYP BM-3 to produce by recombinant DNAtechniques muteins which have modifications at particular amino acidpositions compared with the wild-type CYP BM-3.

Muteins which are particularly suitable for the process of the inventionare those having an amino acid other than Ala at position 74, and/orhaving an amino acid other than Phe at position 87, and/or having anamino acid other than Leu at position 188, and/or having an amino acidother than Phe at position 386. The modifications at the statedpositions can be introduced either as single mutations or elsecumulatively as multiple mutations.

Such a mutein, which is particularly suitable because of its widesubstrate range, is the one having the following three amino acidsubstitutions compared with the wild-type CYP BM-3: position 74 Alareplaced by Gly, position 87 Phe replaced by Val, position 188 Leureplaced by Gin. This mutein can be employed in particular for thehydroxylation of alkanes and aromatic compounds.

A further mutein which is particularly suitable for the hydroxylation ofβ-ionone is the one having the following three amino acid substitutionscompared with the wild-type CYP BM-3: position 74 Ala replaced by Glu,position 87 Phe replaced by Val, position 386 Phe replaced by Ser.

The embedding of enzymes in sol-gel matrices is described in a reviewarticle by I. Gill, Chem. Mater. 2001, 13, 3404-3421.

Particularly suitable sol-gel matrices for the process of the inventionare those based on silica. Particularly suitable sol-gel matrices can beprepared from alkoxysilanes, especially from tetraalkoxysilanes, inparticular tetraethoxysilane (TEOS) and tetramethoxysilane (TMOS).Concerning the preparation of the sol-gel matrices and the embedding ofCYP, reference is made to the abovementioned article by Gill (I. Gill,Chem. Mater. 2001, 13, 3404-3421), which is incorporated herein byreference.

The enzymatic NADPH-regenerating system (called NADPH-RS hereinafter)consists of an NAD⁺- or NADP⁺-dependent enzyme which oxidizes asubstrate to a product with simultaneous reduction of the NAD⁺ to NADHor of the NADP⁺ to NADPH. Suitable in particular for this purpose areall NAD⁺- or NADP⁺-dependent dehydrogenases, especially microbialdehydrogenases and especially formate dehydrogenases.

A preferred embodiment of the NADPH-RS is the NADP⁺-dependent formatedehydrogenase (EC 1.2.1.2), abbreviated to FDH hereinafter, fromPseudomonas sp. 101. FDH catalyzes the NAD⁺ dependent oxidation offormate to CO₂. The advantages of this system are the low substratecosts (formate) and the ease of removal of the resulting product (CO₂).A mutated form of FDH has a high activity with NADP⁺ and can thereforebe employed particularly well as NADPH-regenerating enzyme incombination with CYP. This particularly suitable mutated form of FDH isdescribed by Tishkov et al. Biotechnol. Bioeng. 1999, 64, 187-193 and bySeelbach et al. Tetrahedron Lett. 1996, 37, 1377-1380. These specifieddocuments describe both the natural and the mutated FDH, and processesfor the preparation thereof, especially recombinant expression thereofin E. coli.

The embedding of CYP and NADPH-RS in a sol-gel matrix can take placesimultaneously or in separate mixtures. Simultaneous embedding meansthat firstly a preparation of CYP, preferably a solution of CYP, iscombined with a preparation of NADPH-RS, preferably a solution ofNADPH-RS, and this combined preparation is then embedded in the sol-gelmatrix.

Separate embedding means that a preparation of CYP, preferably asolution of CYP, is embedded in a sol-gel matrix and, in a secondmixture, a preparation of NADPH-RS, preferably a solution of NADPH-RS,is embedded in a sol-gel matrix, and then these two mixtures arecombined.

Separate embedding is preferably used for the process of the inventionbecause subsequent flexible adjustment of the stoichiometric ratio ofCYP to NADPH-RS is also possible with it.

The invention further relates to preparations which comprise a CYP andan NADPH-RS embedded in a sol-gel matrix. Preparations of this type canbe prepared by the process described above. These preparations have theadvantage that it is possible therewith for hydroxylation catalysts tobe prepared in stable form which can be readily stored, and to beprovided for use for specific hydroxylation reactions.

The invention further relates to processes for the enzymatichydroxylation of a substrate using one of the hydroxylation catalystswhich can be prepared according to the invention. A large number ofclasses of organic compounds are suitable as substrate, in particularlong-chain fatty acids (C12-C20) which are hydroxylated in particular atthe subterminal positions ω-1, ω-2, ω-3, but also short-chain fattyacids (Ost et al. FEBS Lett. 2000, 486, 173-177; Li et al. Biochim.Biophys. Acta 2001, 1545, 114-121), indoles (Li et al. Chemistry 2000,6, 1531-1536), polycyclic aromatic hydrocarbons (Li et al. Appl.Environ. Microbiol. 2001, 67, 5735-5739), alkanes (Appel et al. J.Biotechnol. 2001, 88, 167-171) and styrenes (Li et al. FEBS Lett. 2001,508, 249-252).

Conversion of these substrates with the hydroxylation catalyst preparedaccording to the invention takes place under the conditions known to theskilled worker for enzymatic reactions. The reaction can be carried outin a wide temperature range between 0 and 70, preferably between 5 and50 and particularly preferably between 10 and 40° C.

The substrate to be hydroxylated can be dissolved or suspended in anorganic or aqueous solvent, and with liquid substrates it is possible insome circumstances to dispense entirely with the addition of solvents. Amixture of aqueous and organic solvents, especially DMSO/water, ispreferred for the conversion. DMSO/water mixtures in which DMSO amountsto 1-10% v/v are particularly suitable.

The hydroxylation reaction can be carried out batchwise or continuously.In a preferred embodiment with FDH as NADPH-RS, the reaction is carriedout continuously, with continuous feeding of formate ions, in additionto the substrate to be hydroxylated, into the reaction, and continuousremoval of the hydroxylated product and of the resulting CO₂ from thereaction.

The invention is illustrated further by the following examples.

Activity and Stability of the Sol-Gel Immobilized CYP BM-3

The model reaction used was the hydroxylation of p-nitrophenoxydecanoicacid (10-pNCA) which permits easy photometric detection of thep-nitrophenolate product formed (Schwaneberg et al. Anal. Biochem. 1999,269, 359-366).

The immobilized enzyme forms a cloudy mixture after addition to thereaction mixture. Direct kinetic experiments to determine the activityof the sol-gel-embedded CYP BM-3 were therefore impossible. The activityof the sol-gel-embedded CYP BM-3 was therefore determined by incubatingall the components of the standard pNCA test for a certain time and thencentrifuging in order to remove the yellow reaction product from thesolid catalyst. The absorption at 410 nm was then determined from thesupernatant.

Investigations of the long-term stability revealed that the CYP BM-3embedded in the sol-gel suffered no loss of activity over 36 days at 4°C. The free enzyme had a half-life of 26 days on storage in 50 mM KPiand a half-life of 288 days on stabilization with 50% glycerol. Thehalf-life of the sol-gel-embedded enzyme is considerably longer thanthese 288 days.

The immobilized enzyme of the invention displays remarkably highstability even at 25° C. The half-life at this temperature is determinedto be 29 days.

A further selective hydroxylation reaction was carried out with thesol-gel-embedded CYP BM-3 on the substrate n-octane. 79% of theprecursor were hydroxylated. The regio isomers 2-octanol, 3-octanol and4-octanol were obtained in a molar ratio of 1:2.1:1.6 (detected by gaschromatography).

A further selective hydroxylation reaction was carried out with thesol-gel-embedded CYP BM-3 on the substrate naphthalene. 77% of theprecursor were hydroxylated. The main product obtained was 1-naphthol(85%), and 2-naphthol was obtained as by-product (15%) (detected by gaschromatography).

Cofactor Regeneration

Two possibilities were investigated for the cofactor regenerationaccording to the invention. Co-immobilization of both enzymes (CYP andFDH) was investigated in a first series of experiments, and the twoenzymes were immobilized separately and then mixed in the ratio 1:1(m/m) in a second series of experiments. In both cases, the embeddingtook place in a TEOS sol-gel matrix.

Co-immobilized enzymes from the first experimental series were put intothe p-NCA test with a 10-fold excess of p-NCA over oxidized NADP⁺.

The activity of the separately immobilized FDH in combination withseparately immobilized CYP was considerably higher than with theco-immobilized enzymes.

After a reaction time of three hours, the co-immobilized enzymes showeda conversion of up to 28% of the pNCA, while the mixture of separatelyimmobilized enzymes led to a conversion of up to 75%.

These results make it possible to operate a continuously operatingbioreactor based on a sol-gel-embedded CYP with NADPH-RS.

1. A process for preparing a hydroxylation catalyst by i) embedding acytochrome P450 monooxygenase in a sol-gel matrix, ii) embedding anenzymatic NADPH-regenerating system in a sol-gel matrix, and combiningthe two components i) and ii) unless they were already mixed togetherbefore the embedding.
 2. A process as claimed in claim 1, whereinenzymes which had originally been isolated from the genus Bacillus areused as monooxygenases.
 3. A process as claimed in claim 2, wherein themonooxygenases have been isolated from Bacillus megaterium.
 4. Apreparation comprising a cytochrome P450 monooxygenase and an enzymaticNADPH-regenerating system in a sol-gel matrix.
 5. A process for theenzymatic hydroxylation of a substrate by reacting the substrate with ahydroxylation catalyst which can be prepared as claimed in claim
 1. 6. Aprocess as claimed in claim 5, wherein β-ionone is employed assubstrate.